PROJECT SUMMARY Limb loss is a major health concern affecting nearly two million patients in the U.S. Current therapeutic approaches are mainly limited to prosthetic limbs, but despite increasing sophistication, they are far from perfect replacements. The ideal therapy would be to stimulate the patient?s remaining tissues to mount a regenerative response. Because so little is known about why human limbs do not spontaneously regenerate, while limbs in other species can, the goal of stimulating human limb regeneration is currently quite futuristic. Advances in understanding how some highly-regenerative species, such as axolotl salamanders, regenerate entire limbs are beginning to provide important molecular insights into how a complex, multi-tissue limb can be regenerated. However, as these animals are quite diverged from humans, the translation of these findings into the human forum remains murky. A concerted effort is needed to create a research pipeline to leverage new data from axolotls and bring it into a testable experimental context in a mammal. The ideal mammal to use as a bridge is the mouse since a robust research pipeline connecting mouse and human already exists and since there are numerous genetic tools in mice that could be transformative for these questions. While mice cannot regenerate full limbs, they can regenerate the distal tips of their digits, similar to young humans. The process of mouse digit tip regeneration appears to share some similarities with full limb regeneration in axolotls; for example, mouse digit tip regeneration relies on lineage-restricted progenitor cells, and for most lineages, the same has been found in axolotls. Furthermore, both utilize a blastema, where activated progenitors accumulate at the tip of the stump, to facilitate regeneration. Yet, there is a paucity of scientific understanding of how connected these two systems may be at a molecular level. Here, we propose to establish a formal means of translating limb regeneration findings from axolotl to mouse. We will directly test a factor we recently uncovered as an antagonist of limb regeneration in axolotls also operates to antagonize mouse digit tip regeneration. In addition, using regenerating mouse digit tips, we will screen for expression of genes identified in axolotl as candidate factors for supporting blastema functions. Using an ex vivo embryonic mouse digit tip regeneration assay, we test the necessity of 10 signaling pathways identified in the axolotl studies as pro-regenerative. Together, performing these experiments and establishing these assays will advance both the understanding of how the two systems may be connected at the molecular level, and bring insights from basic scientific studies closer to therapeutic approaches for patients.